Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.     

Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.     

Essential roles of IkappaB kinases alpha and beta in serum- and IL-1-induced human VSMC proliferation

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-kappaB (NF-kappaB) following phosphorylation of its inhibitory subunit (IkappaB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IkappaB kinases (IKKs) and NF-kappaB in both IL-1- and serum-induced VSMC proliferation. IL-1beta (1 ng/ml) induced marked and persistent NF-kappaB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24-96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-kappaB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKalpha or IKKbeta, in which K(44) was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Although serum activated NF-kappaB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKalpha (K44A) and IKKbeta (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.     

Caspase inhibition sensitizes inhibitor of NF-kappaB kinase beta-deficient fibroblasts to caspase-independent cell death via the generation of reactive oxygen species

Cells lacking functional NF-kappaB die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappaB-dependent anti-apoptotic genes. NF-kappaB activation requires the IkappaB kinase (IKK) complex containing two catalytic subunits named IKKalpha and IKKbeta that regulate distinct NF-kappaB pathways. IKKbeta is critical for classical signaling that induces pro-inflammatory and anti-apoptotic gene profiles, whereas IKKalpha regulates the non-canonical pathway involved in lymphoid organogenesis and B-cell development. To determine whether IKKalpha and IKKbeta differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-beta receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKKalpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKKalpha functions in this process. Surprisingly, death of IKKbeta-/- MEFs was not blocked by caspase inhibition, demonstrating that IKKbeta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKKbeta-/- MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti-oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKKalpha and IKKbeta regulate cytokine-induced apoptosis, and IKKbeta additionally represses reactive oxygen species- and mitochondrial-dependent CICD.     

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.     

Signaling by the Mpl receptor involves IKK and NF-kappaB

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.     

Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.     

Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.     

AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Cyclooxygenase-2 (COX-2) is a crucial mediator in mechanically induced bone formation. AMP-activated protein kinase (AMPK) has reported to sense and regulate the cellular energy status in various cell types. Here we found that US-mediated COX-2 expression was attenuated by LKB1 and AMPKalpha1 small interference RNA (siRNA) in human osteoblasts. Pretreatment of osteoblasts with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide also inhibited the potentiating action of US. US increased the kinase activity and phosphorylation of LKB1, AMPK and p38. Stimulation of osteoblasts with US activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKKalpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by araA, SB203580 and LKB1 siRNA. Our results suggest that US increased COX-2 expression in osteoblasts via the LKB1/AMPKalpha1/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Ultrasound induces cyclooxygenase-2 expression through integrin, integrin-linked kinase, Akt, NF-kappaB and p300 pathway in human chondrocytes

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the precise molecular events generated by US in chondrocytes have not been clarified well. Here we found that US stimulation transiently increased the surface expression of alpha2, alpha5, beta1 or beta3 but not alpha3 or alpha4 integrins in human chondrocytes, as shown by flow cytometric analysis. US stimulation increased prostaglandin E(2) formation as well as the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin beta1 and beta3 antibodies or beta1 and beta3 integrin small interference RNA attenuated the US-induced COX-2 expression. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited the potentiating action of US. US stimulation promotes kinase activity of ILK, phosphorylation of Akt. In addition, US stimulation also induces IKKalpha/beta phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the COX-2 promoter was enhanced by US. Taken together, our results provide evidence that US stimulation increases COX-2 expression in chondrocytes via the integrin/ILK/Akt/NF-kappaB and p300 signaling pathway.     

IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.